Nucleic acid testing (NAT) can be used to diagnose infectious diseases by identifying the pathogen's genetic material. NAT is usually performed in centralized laboratories by highly trained personnel on large, complex, expensive equipment. However, testing in a central laboratory is not ideal for applications that require a rapid answer to facilitate treatment and improve patient outcomes. Additionally, in developing countries, the diagnosis of endemic infectious diseases through NAT in central laboratories is hampered by the lack of suitable facilities, trained personnel, and logistics chains.
Nucleic acid amplification testing (NAAT) enables sensitive and specific diagnosis of infectious diseases, producing accurate results in less than one day. However, most NAAT technologies require additional time for samples to be transferred to a central laboratory with results transferred back to the care provider. Point of care (POC) NAAT eliminates these often substantial additional delays and enables testing and treatment initiation in the same visit, which is more time efficient and may reduce the risk of losing a patient to delayed follow-up.
PCR-based fully or partially integrated NAAT systems for infectious disease diagnosis are in development or on the market, but all of these PCR-based systems are relatively expensive bench-top systems due to the complexity associated with thermocycling and real time fluorescence detection.
NAAT for infectious diseases requires sample input volumes to reach the required limit of detection (LOD)—which, in general, range from 100 μL to several mL. After sample preparation, the purified and concentrated DNA is combined with additional reagents, resulting in a master-mix volume between 30-100 μL which is then processed for amplification and detection. Mesofluidic systems are capable of processing samples in this volume range, including up to several hundred microliters. Since the attainable LOD scales linearly with sample input volume, this volume restriction places microfluidic systems (e.g., PCR systems) at a significant disadvantage. Moreover, real time PCR requires thermocycling and fluorescence optics for detection, and the fluorescence optics are difficult to implement in a compact, low cost, and robust instrument for use in low-resource settings. In contrast to PCR, isothermal NAAT requires a single reaction temperature, and therefore utilizes more simplified instrumentation than a PCR system.
Lateral flow devices work well for POC diagnostics because lateral flow devices can be manufactured inexpensively in large quantities, rely on passive fluidics, and provide a clear visual readout without additional instruments. While progress has been made in automating isothermal nucleic acid amplification, to date, users must perform sample preparation and amplification in separate manual steps, and then transfer the tube with the amplified master-mix for lateral flow detection. As such, there are currently no handheld inexpensive systems that automate and integrate isothermal nucleic acid amplification and lateral flow detection. Accordingly, a need exists for a low-cost, disposable cartridge in conjunction with a compact, inexpensive device that contains low power electronics, and is capable of processing a sample for nucleic acid amplification with lateral flow detection.